菌丝霉素的高效表达、纯化及活性  

High-level Expression,Purification and Activity of the Plectasin

作　　者：梁文[1] 胡又佳[1] 朱宝泉[1] 谢丽萍[1] 

机构地区：[1]中国医药工业研究总院上海医药工业研究院,创新药物与制药工艺国家重点实验室,上海200040

出　　处：《中国医药工业杂志》2013年第1期21-26,共6页Chinese Journal of Pharmaceuticals

基　　金：上海市启明星(11QB1406400);国家科技部973计划(2010CB735601);中国国药集团开发基金资助计划项目资助(2011HY19)

摘　　要：将来自腐生子囊菌中的菌丝霉素成熟肽基因经大肠杆菌密码子优化后,克隆到pET32a(+)载体中,构建了含His-tag标签和TEV酶识别位点的菌丝霉素表达质粒pYG330,使含菌丝霉素的融合蛋白在大肠杆菌中以可溶形式高效表达。建立了菌丝霉素的纯化路线,将融合蛋白进行TEV酶切后脱盐再进行亲和色谱分离,收集目的蛋白冻干浓缩,通过高效液相色谱进一步纯化获得纯度72%的目的多肽。纯化所得菌丝霉素对临床耐药金黄色葡萄球菌M-2和枯草芽孢杆菌有明显的抑菌活性。The gene coding plectasin mature peptide isolated from a saprophytic fungus was optimized according to the E. coli codon preference, and inserted into a pET-32a （＋）-TEV carrier. Then a recombinant plasmid pYG330 for high-level expression of recombinant plectasin fusion protein was constructed. Fusion protein were expressed in the soluble fraction. The fusion protein contained both His-tag label for affinity chromatography and specific enzymes recognition sites of TEV enzyme for fusion protein efficient cutting. High purity plectasin was achieved through three key purification processes, affinity chromatography after desalting, enrichment by freeze-dry, and another step of HPLC. Plectasin obtained in this study showed marked antibacterial activity against clinical drug-resistance bacteria of Staphylococcus aureus M-2 and Bacillus subtilis.

关 键 词：菌丝霉素 高效表达 分离 纯化 抑菌活性 临床耐药菌 

分 类 号：TQ465.92[化学工程—制药化工]